LOCUS       sequence-134396-        4754 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mRuby2_NLSPUFa in pCR8 gateway donor vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4754)
  AUTHORS   Cheng AW, Jillette N, Lee P, Plaskon D, Fujiwara Y, Wang W, 
            Taghbalout A, Wang H
  TITLE     Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation 
            and genomic labeling.
  JOURNAL   Cell Res. 2016 Feb;26(2):254-7. doi: 10.1038/cr.2016.3. Epub 2016 
            Jan 15.
  PUBMED    26768771
REFERENCE   2  (bases 1 to 4754)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/cr.2016.3";
            journalName: "Cell Res"; date: "2016-02"; volume: "26"; issue: "2"; 
            pages: "254-7"
COMMENT     Sequence Label: pAC1404-pCR8-mRuby2_NLSPUFa
FEATURES             Location/Qualifiers
     source          1..4754
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      686..695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             707..724
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             728..760
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(764..772,773..787)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             794..1504
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             1541..1561
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2633..2732)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2749..2768)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2750..2768)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2773..2789)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2773..2789)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3220..4011
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      4104..4692
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4593..4612
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gaattcaccg gtgccgccac catggtgcgg ggttctcatc atcatcatca
      721 tcatggtatg gctagcatga ctggtggaca gcaaatgggt cgggatctgt acgacgatga
      781 cgataaggat ccgatggtgt ctaagggcga agagctgatc aaggaaaata tgcgtatgaa
      841 ggtggtcatg gaaggttcgg tcaacggcca ccaattcaaa tgcacaggtg aaggagaagg
      901 caatccgtac atgggaactc aaaccatgag gatcaaagtc atcgagggag gacccctgcc
      961 atttgccttt gacattcttg ccacgtcgtt catgtatggc agccgtactt ttatcaagta
     1021 cccgaaaggc attcctgatt tctttaaaca gtcctttcct gagggtttta cttgggaaag
     1081 agttacgaga tacgaagatg gtggagtcgt caccgtcatg caggacacca gccttgagga
     1141 tggctgtctc gtttaccacg tccaagtcag aggggtaaac tttccctcca atggtcccgt
     1201 gatgcagaag aagaccaagg gttgggagcc taatacagag atgatgtatc cagcagatgg
     1261 tggtctgagg ggatacactc atatggcact gaaagttgat ggtggtggcc atctgtcttg
     1321 ctctttcgta acaacttaca ggtcaaaaaa gaccgtcggg aacatcaaga tgcccggtat
     1381 ccatgccgtt gatcaccgcc tggaaaggtt agaggaaagt gacaatgaaa tgttcgtagt
     1441 acaacgcgaa cacgcagttg ccaagttcgc cgggcttggt ggtgggatgg acgagctgta
     1501 caagggaggc ggcggtagcg ggccggccgg gatcctcccc cccaagaaaa agaggaaggt
     1561 atctagaggc cgcagccgcc ttttggaaga ttttcgaaac aaccggtacc ccaatttaca
     1621 actgcgggag attgctggac atataatgga attttcccaa gaccagcatg ggtccagatt
     1681 cattcagctg aaactggagc gtgccacacc agctgagcgc cagcttgtct tcaatgaaat
     1741 cctccaggct gcctaccaac tcatggtgga tgtgtttggt aattacgtca ttcagaagtt
     1801 ctttgaattt ggcagtcttg aacagaagct ggctttggca gaacggattc gaggccacgt
     1861 cctgtcattg gcactacaga tgtatggcag ccgtgttatc gagaaagctc ttgagtttat
     1921 tccttcagac cagcagaatg agatggttcg ggaactagat ggccatgtct tgaagtgtgt
     1981 gaaagatcag aatggcaatc acgtggttca gaaatgcatt gaatgtgtac agccccagtc
     2041 tttgcaattt atcatcgatg cgtttaaggg acaggtattt gccttatcca cacatcctta
     2101 tggctgccga gtgattcaga gaatcctgga gcactgtctc cctgaccaga cactccctat
     2161 tttagaggag cttcaccagc acacagagca gctggtacag gatcaatatg gaaattatgt
     2221 aatccaacat gtactggagc acggtcgtcc tgaggataaa agcaaaattg tagcagaaat
     2281 ccgaggcaat gtacttgtat tgagtcagca caaatttgca agcaatgttg tggagaagtg
     2341 tgttactcac gcctcacgta cggagcgcgc tgtgctcatc gatgaggtgt gcaccatgaa
     2401 cgacggtccc cacagtgcct tatacaccat gatgaaggac cagtatgcca actacgtggt
     2461 ccagaagatg attgacgtgg cggagccagg ccagcggaag atcgtcatgc ataagatccg
     2521 gccccacatc gcaactcttc gtaagtacac ctatggcaag cacattctgg ccaagctgga
     2581 gaagtactac atgaagaacg gtgttgactt agggtgatta attaagaatt cgacccagct
     2641 ttcttgtaca aagttggcat tataaaaaat aattgctcat caatttgttg caacgaacag
     2701 gtcactatca gtcaaaataa aatcattatt tgccatccag ctgatatccc ctatagtgag
     2761 tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
     2821 tgttacattg cacaagataa aaatatatca tcatgcctcc tctagaccag ccaggacaga
     2881 aatgcctcga cttcgctgct gcccaaggtt gccgggtgac gcacaccgtg gaaacggatg
     2941 aaggcacgaa cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt
     3001 atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg
     3061 gcggttttca tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc
     3121 aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt
     3181 acgcagcagg gcagtcgccc taaaacaaag ttaaacatca tgagggaagc ggtgatcgcc
     3241 gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg
     3301 ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat
     3361 attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc
     3421 aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa
     3481 gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg
     3541 caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc
     3601 gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt
     3661 ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat
     3721 gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg
     3781 cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc
     3841 gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct
     3901 agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa
     3961 gaatttgtcc actacgtgaa aggcgagatc accaaggtag tcggcaaata accctcgagc
     4021 cacccatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc
     4081 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4141 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4201 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     4261 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4321 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4381 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4441 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagcattga
     4501 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4561 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4621 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4681 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4741 tttgctcaca tgtt
//
